A separation technology called supercritical fluid chromatography (SFC) has advanced over the past decade. SFC uses highly compressible mobile phases, which typically employ carbon dioxide (CO2) as a principle component. In addition to CO2, the mobile phase frequently contains an organic solvent modifier, which adjusts the polarity of the mobile phase for optimum chromatographic performance. A common gradient range for gradient SFC methods might occur in the range of 2% to 60% composition of the organic modifier. Since different components of a sample may require different levels of organic modifier to elute rapidly, a common technique is to continuously vary the mobile phase composition by linearly increasing the organic modifier content. This technique is called gradient elution. SFC instruments, while designed to operate in regions of temperature and pressure above the critical point of carbon dioxide (CO2), are typically not restricted from operation well below the critical point. In this lower region, especially when organic modifiers are used, chromatographic behavior remains superior to traditional HPLC and often cannot be distinguished from true supercritical operation.
SFC has been proven to have superior speed and resolving power compared to traditional methods, such as high-performance liquid chromatography (HPLC) for analytical applications. This results from the dramatically improved diffusion rates of solutes in SFC mobile phases compared to other analytical methods, such as HPLC mobile phases. Separations have been accomplished as much as an order of magnitude faster using SFC instruments compared to HPLC instruments using the same chromatographic column. A key factor to optimizing SFC separations is the ability to independently control flow, density and composition of the mobile phase over the course of the separation. SFC instruments used with gradient elution also re-equilibrate much more rapidly than corresponding HPLC systems. As a result, they are ready for processing a consecutive sample after a shorter period of time.
Analysts have several objectives in employing preparative elution chromatography. First, they wish to achieve the highest available purity of each component of interest. Second, they wish to recover the maximum amount of the components of interest. Third, they wish to process sequential, possibly unrelated samples as quickly as possible and without contamination from prior samples. Finally, it is frequently desirable to recover samples in a form that is rapidly convertible either to the pure, solvent-free component or to a solution of known composition which may or may not include the original collection solvent.
SFC systems operate with varying compositions of two independently controlled flow streams: the first flow stream delivers a highly compressed fluid, such as carbon dioxide, and a second stream delivers a modifier solvent, such as methanol. The two independent flow streams are combined into a single flow stream that enters the separation column. In both HPLC and SFC, sample is normally introduced into the flow stream by means of an injection valve. Common injection valves are fixed-loop multi-port injection valves with either internal or external loops. Direct full loop injections are normal means of sample introduction in SFC so that a packed column has similar quantitative reproducibility to LC using fixed-loop injectors. Injection valves used in SFC sample introduction present special hazards caused by the higher pressures (up to 600 bar) found in SFC systems. Sample may be manually injected into the sample loop with a syringe through a fill port.
In SFC systems, the mobile phase is typically a mixture of an organic modifier and a highly compressed fluid, such as carbon dioxide (CO2) delivered by two independent pumps. In the prior art, samples are dissolved in an organic solvent and then injected into the mobile phase stream at a location 21 just prior to entering a separation column. A problem with this method is that the organic modifier is typically the strongest solvent in the system and can cause uncontrolled elution of the sample components well into the bed of the separation column before the controlled process of gradient elution becomes effective. In this configuration, the liquid phase sample may never completely mix with the mobile phase in the SFC flow stream, thereby causing a concentrated, separated slug of sample to enter a separation column and cause smearing, high background noise, or varying separation times. The resultant component peaks will experience substantial broadening, producing poor chromatographic results. Particularly, the problem occurs with poorly retained components, which experience the greatest degree of broadening and are typically the most difficult to separate. The problems with broadening of peaks becomes severely and multiplied when larger sample volumes are used to apply the sample components to the column 26.
At least one prior art system has been built to inject samples into the modifier flow stream prior to mixing with the compressible fluid (CO2) high-pressure flow stream in an SFC system is from Prochrom International, Champigneulles, France. The system is designed for sample injection for SFC into the modifier stream that uses a pump to continuously circulate sample in an external flow path having a plurality of control valves to control flow direction. Such a system has up to eight control valves that are manipulated to direct the sample into the flow stream. The pump circulates sample from a reservoir until flow loop contents are ready for injection into the low pressure flow stream. To inject, valves are closed to the reservoir and circulation pump and other valves are opened to the flow stream, thereby injecting the sample volume by flushing out the sample through the flow path containing sample with the moving flow stream. After injection, valves to the flow stream are closed and valves to the circulation pump are re-opened.
A few of the draw backs associated with the Prochrom configuration are that a pump must continually pump sample through a looping flow path and that a plurality of valves must be manually or automatically manipulated to make an injection. This time-consuming method is not amenable to the rapid repeated injections available in SFC and also add to complication and inaccuracies of injection. Also, since sample is continuously pumped through the flow loop prior to injection, partial loop injections are not possible. After injection, part of the flow path containing sample can become filled with flow stream contents, which in turn is circulated into the sample reservoir by the pump, thereby diluting the sample reservoir and subsequent sample injections to an unknown degree.
There is a need for efficient premixing samples with the actual mobile phase, prior to injection, to apply samples to a separation column in supercritical fluid chromatography or high performance liquid chromatography. A method for injection must be timely and not bottleneck a rapid injection, separation, and collection process in an SFC system. The method should control how the sample is delivered to the separation column. Finally, a method for premixing should be environmentally friendly and generate as little hazardous chemicals for disposal as possible and prevent hazardous chemicals from escaping into the atmosphere.
The invention is a method for introducing sample into the flow stream of chromatography systems that operate at high pressures, such as high-performance liquid chromatography (HPLC) or supercritical fluid chromatography (SFC), and mix two separate flow streams to create a single mobile phase flow stream. The method of the preferred embodiment is an improvement to high-pressure chromatography systems by introducing samples into a low-pressure modifier flow stream at a point prior to mixing with a high-pressure compressible fluid flow stream.
The present invention uses a multi-port injection valve with a sample injection loop to inject into the modifier flow stream. A syringe or syringe pump adds sample into the loop between injections. No additional valves or circulating pumps are necessary. Partial-loop injections into the flow stream are also possible. Because of the precise nature of the injections and no continual circulation of the sample into and out of a sample flow stream, the possibility of dilution of an injected sample is minimal. Virtually all of the sample injected into the modifier flow stream reaches the separation column.
The preferred method provides greater control over the SFC process by providing for pre-mixing of sample into a low-pressure flow stream and balancing of high-pressure compressible and low pressure incompressible flow streams to enhance the speed of separation and quality of chromatographic results. Focusing of sample components at the column head is also controlled by changing the ratio of compressible to incompressible flow streams.
The present invention addresses safety and time delay problems caused by injections of sample into high-pressure mobile phase flow streams. A sample injection valve in SFC introduces a measured sample into the mobile phase flow stream prior to entering a chromatography column. During an injection, the valve loop discharges sample contents into a mobile phase flow stream. After injection, high-pressure mobile phase becomes trapped inside the sample loop when the valve is returned to a load position. Setting the injection valve sample loop back to a load position also exposes the sample loop that is under high pressure to atmospheric pressure inside the laboratory. Compressed mobile phase in SFC will rapidly expand approximately 500 times when exposed to atmospheric pressure. A hazardous condition occurs when mobile phase trapped in an injection loop explodes out of the system through a sample fill port connected to the loop, and out into open air. Also, by moving the injection location to a low-pressure flow stream, blowback of high-pressure mobile phase from an injection loop is avoided.
Dissipating pressure from a sample loop to a waste line causes delays to the injection process which slows the entire SFC sample processing time. The injection valve location that is positioned upstream of the mixing point for the incompressible and compressible (or supercritical) fluid flow streams removes the time-delays associated with dissipating pressures trapped in an injection valve and blow-back from an injection valve into a laboratory. The modifier flow stream at this location has lower pressure. Therefore, time delays caused by waiting for dissipation of a pressure-charged injection valves are avoided.